The inventor discovered a new enzyme in alkaliphilic Bacillus sp. N16-5 (Bacillus sp. N16-5), which has several activities, including: (a) the activity of using HMBPP as a substrate to produce DMAPP and IPP; (b) the activity of using HMBPP as a substrate to produce isoprene; and (c) the activity of using DMAPP as a substrate to produce isoamylene (2-methyl-2-butene and 3-methyl-1-butene). Furthermore, the inventor obtained two mutants (H131N and E133Q) by modifying the enzyme. The two mutants lose the activity of using HMBPP as a substrate to produce DMAPP and IPP and the activity of producing isoamylene, but retain the activity of using HMBPP as a substrate to produce isoprene, and have a stronger capability of producing isoprene in a cell than that of the wild-type enzyme (having the yield of isoprene increased by 3-4 folds). On the basis of the above, the inventor develops new methods for synthesis of isoprene and isoamylene.
Therefore, in one aspect, the invention provides a polypeptide, which has an activity of using 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) as a substrate to produce isoprene, and has an amino acid sequence selected from the group consisting of:
(1) an amino acid sequence set forth in SEQ ID NO: 2;
(2) an amino acid sequence having an identity of at least 90% with SEQ ID NO: 2; and
(3) an amino acid sequence that differs from SEQ ID NO: 2 by substitution, deletion or addition of one or more amino acid residues.
In some preferred embodiments, the polypeptide according to the invention has an amino acid sequence having an identity of at least 90%, preferably an identity of at least 91%, an identity of at least 92%, an identity of at least 93%, an identity of at least 94%, an identity of at least 95%, an identity of at least 96%, an identity of at least 97%, an identity of at least 98%, or an identity of at least 99% with SEQ ID NO: 2.
In some preferred embodiments, the polypeptide according to the invention has an amino acid sequence that differs from SEQ ID NO: 2 by substitution, deletion or addition of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid residues.
In some preferred embodiments, the polypeptide according to the invention has an amino acid sequence that differs from SEQ ID NO: 2 by conservative substitution of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid residues.
In some preferred embodiments, the polypeptide according to the invention has an amino acid sequence that is SEQ ID NO: 2 or differs from SEQ ID NO: 2 by substitution of one or more (e.g., 1) amino acid residues. For example, in some preferred embodiments, the polypeptide according to the invention has an amino acid sequence that differs from SEQ ID NO: 2 by amino acid substitution at position 131 or 133 of SEQ ID NO: 2. In some preferred embodiments, the polypeptide according to the invention has an amino acid sequence that differs from SEQ ID NO: 2 by mutation of histidine to asparagine at position 131 of SEQ ID NO: 2, or by mutation of glutamic acid to glutamine at position 133 of SEQ ID NO: 2.
In some preferred embodiments, the polypeptide according to the invention has an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3 and 4.
In some preferred embodiments, the polypeptide according to the invention has an activity of using dimethylallyl pyrophosphate (DMAPP) as a substrate to produce 2-methyl-2-butene and 3-methyl-1-butene.
In some preferred embodiments, the polypeptide according to the invention does not have an activity of using dimethylallyl pyrophosphate (DMAPP) as a substrate to produce 2-methyl-2-butene and 3-methyl-1-butene.
In another aspect, the invention provides an isolated nucleic acid, encoding the polypeptide as described above. In another aspect, the invention provides a vector, comprising the isolated nucleic acid. In some preferred embodiments, the isolated nucleic acid according to the invention encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3 and 4. Vectors useful for inserting a polynucleotide of interest are well known in the art. In an embodiment, the vector, for example, is a plasmid, a cosmid, a phage, etc.
In another aspect, the invention further relates to a host cell comprising the isolated nucleic acid or the vector. The host cell includes, but is not limited to a prokaryotic cell such as E. coli cell and bacillus cell (e.g., Bacillus alcalophilus, Bacillus subtilis), and a eukaryotic cell such as a yeast cell, an insect cell, a plant cell and an animal cell.
In some preferred embodiments, the isolated nucleic acid is heterogenous relative to the cell. In some preferred embodiments, the isolated nucleic acid is exogenous relative to the cell.
In some preferred embodiments, the cell further comprises a nucleic acid encoding an electron transporter (e.g., ferredoxin) and/or an enzyme (e.g., ferredoxin reductase) needed for an electron transporter to transport electron, or expresses an electron transporter (e.g., ferredoxin) and/or an enzyme (e.g., ferredoxin reductase) needed for an electron transporter to transport electron. For example, the cell may further comprise a nucleic acid encoding ferredoxin and ferredoxin reductase, or express ferredoxin and ferredoxin reductase. In some preferred embodiments, the electron transporter and/or the enzyme needed for an electron transporter to transport electron is endogenous relative to the cell. In some preferred embodiments, the electron transporter and/or the enzyme needed for an electron transporter to transport electron is exogenous relative to the cell. For example, the ferredoxin and/or ferredoxin reductase is endogenous or exogenous relative to the cell. In some preferred embodiments, the cell further comprises an exogenously introduced nucleic acid encoding ferredoxin, and/or an exogenously introduced nucleic acid encoding ferredoxin reductase. In some preferred embodiments, the ferredoxin reductase is ferredoxin-NADP+ reductase (EC 1.18.1.2).
In some preferred embodiments, the cell further expresses a polypeptide of DXP pathway. Preferably, the polypeptide of DXP pathway is selected from the group consisting of 1-deoxy-D-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR; EC 1.1.1.267), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (MCT; EC 2.7.7.60), 4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol kinase (CMK; EC 2.7.5.148), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS; EC 4.6.1.12), 4-hydroxy-3-methyl-2-(E)-butenyl-diphosphate synthase (IspG; EC 1.17.7.1), and any combination thereof. In some preferred embodiments, the polypeptide of DXP pathway is endogenous relative to the cell. In some preferred embodiments, the polypeptide of DXP pathway is exogenous relative to the cell.
In some preferred embodiments, the cell further expresses isoprene synthase (IspS; EC 4.2.3.27). In some preferred embodiments, the cell does not express isoprene synthase (IspS; EC 4.2.3.27).
In some preferred embodiments, the cell is a prokaryotic cell, such as E. coli or Bacillus spp. or blue-green algae.
In another aspect, the invention further relates to a composition, comprising the polypeptide according to the invention, HMBPP, NADPH or NADH, an electron transporter (e.g., ferredoxin), and an enzyme (e.g., ferredoxin reductase) needed for an electron transporter to transport electron. In some preferred embodiments, the composition comprises the polypeptide, HMBPP, NADPH, ferredoxin and ferredoxin reductase (e.g., ferredoxin-NADP+ reductase). The composition according to the invention is useful in synthesis of isoprene in vitro.
In another aspect, the invention further relates to a composition, comprising a polypeptide having an amino acid sequence set forth in SEQ ID NO: 2, DMAPP, NADPH or NADH, an electron transporter (e.g., ferredoxin), and an enzyme (e.g., ferredoxin reductase) needed for an electron transporter to transport electron. In some preferred embodiments, the composition comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO: 2, DMAPP, NADPH, ferredoxin and ferredoxin reductase (e.g., ferredoxin-NADP+ reductase). The composition according to the invention is useful in synthesis of isoamylene (e.g., 3-methyl-1-butene and/or 2-methyl-2-butene) in vitro.
In another aspect, the invention provides a method for producing isoprene, comprising using the polypeptide according to the invention to convert HMBPP to isoprene.
In some preferred embodiments, the method comprises (a) mixing and incubating (preferably incubating at 20-40° C., e.g., incubating at room temperature or 37° C.) the polypeptide, HMBPP, NADPH or NADH, an electron transporter (e.g., ferredoxin), and an enzyme (e.g., ferredoxin reductase) needed for an electron transporter to transport electron; and (b) collecting isoprene produced in step (a). In some preferred embodiments, in step (a), the polypeptide, HMBPP, NADPH, ferredoxin and ferredoxin reductase (e.g., ferredoxin-NADP+ reductase) are mixed and incubated, for example, incubated at 20-40° C. (e.g., incubated at room temperature or 37° C.), to produce isoprene. In some preferred embodiments, the polypeptide has an amino acid sequence set forth in SEQ ID NO: 3 or 4.
In some preferred embodiments, the method does not involve use of isoprene synthase (IspS: EC 4.2.3.27). In some preferred embodiments, the method is used in production of isoprene in vitro.
In another aspect, the invention provides a method for producing isoprene, comprising (a) culturing a cell expressing the polypeptide according to the invention which is exogenously introduced; and (b) collecting isoprene produced in step (a).
In some preferred embodiments, in step (a), a cell is cultured under conditions suitable, for producing isoprene. For example, in order to promote isoprene production in a cell, the cell may be provided with one or more of the following substances: (1) a culture medium for retaining or promoting cell growth; (2) the substrate of the polypeptide according to the invention, HMBPP; (3) an electron transporter (e.g., ferredoxin); (4) an enzyme (e.g., ferredoxin reductase) needed for an electron transporter to transport electron: and (5) NADPH or NADH.
A variety of suitable culture media for culturing cells are well known by a person skilled in the art, and are commercially available.
In some preferred embodiments, the cell further expresses an electron transporter (e.g., ferredoxin) and/or an enzyme (e.g., ferredoxin reductase) needed for an electron transporter to transport electron. For example, the cell further expresses ferredoxin and ferredoxin reductase. In some preferred embodiments, the electron transporter and/or the enzyme needed for an electron transporter to transport electron is endogenous relative to the cell. In some preferred embodiments, the electron transporter and/or the enzyme needed for an electron transporter to transport electron is exogenous relative to the cell. For example, the ferredoxin and/or ferredoxin reductase may be endogenous or exogenous relative to the cell, in some preferred embodiments, the ferredoxin reductase is ferredoxin-NADP+ reductase (EC 1.18.1.2).
In some cases, the cell naturally expresses ferredoxin and ferredoxin reductase. In such a cell, if is not necessary to exogenously introduce a nucleic acid encoding ferredoxin and ferredoxin reductase. However, it is particularly preferred that in such a cell, a nucleic acid encoding ferredoxin and ferredoxin reductase is further introduced, to increase the expression of ferredoxin and ferredoxin reductase, thereby further enhancing the activity of the polypeptide according to the invention. In some cases, the cell does not express ferredoxin and ferredoxin reductase. In such a cell, it is particularly preferred that a nucleic acid encoding ferredoxin and ferredoxin reductase is introduced, so as to provide a high-efficient electron donor for the polypeptide according to the invention.
In some preferred embodiments, HMBPP may be added to a cell culture medium, so as to provide a substrate for the polypeptide according to the invention. In some preferred embodiments, DXP pathway is established in a cell to promote HMBPP synthesis in the cell, thereby providing a substrate for the polypeptide according to the invention. For example, one or more polypeptides involved in DXP pathway can be expressed in a cell so as to promote. HMBPP synthesis in the cell, thereby providing a substrate for the polypeptide according to the invention.
Therefore, in some preferred embodiments, the cell further expresses a polypeptide of DXP pathway. In some preferred embodiments, the polypeptide of DXP pathway is selected from the group consisting of 1-deoxy-D-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR; EC 1.1.1.267), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (MCT; EC 2.7.7.60), 4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol kinase (CMK; EC 2.7.1.148), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS; EC 4.6.1.12), 4-hydroxy-3-methyl-2-(E)-butenyl-diphosphate synthase (IspG; EC 1.17.7.1), and any combination thereof. In some preferred embodiments, the cell expresses one, two, three, four, five or six of said polypeptides of DXP pathways.
In some preferred embodiments, the polypeptide of DXP pathway is endogenous relative to the cell. In some preferred embodiments, the polypeptide of DXP pathway is exogenous relative to the cell.
In some preferred embodiments, the cell further expresses isoprene synthase (IspS; EC 4.2.3.27), In such a cell, isoprene may be biosynthesized by several pathways. In some preferred embodiments, the cell does not express isoprene synthase (IspS; EC 4.2.3.27).
In some preferred embodiments, the cell is selected from the group consisting of a prokaryotic cell such as E. coli cell and bacillus cell (e.g., Bacillus alcalophilus, Bacillus subtilis), and a eukaryotic cell such as a yeast cell, an insect cell, a plant cell and an animal cell. However, it is particularly preferred that the cell is a prokaryotic cell, such as E. coli or Bacillus spp. or blue-green algae.
In some preferred embodiments, the method is used in the biosynthesis of isoprene.
In another aspect, the invention provides a method for producing isoamylene (e.g., 3-methyl-1-butene and/or 2-methyl-2-butene), comprising using a polypeptide having an amino acid sequence set forth in SEQ ID NO: 2, to convert DMAPP to isoamylene.
In some preferred embodiments, the method comprises (a) mixing and incubating the polypeptide, DMAPP, NADPH or NADH, an electron transporter (e.g., ferredoxin) and an enzyme (e.g., ferredoxin reductase) needed for an electron transporter to transport electron; and (b) collecting isoamylene produced in step (a). In some preferred embodiments, in step (a), the polypeptide, DMAPP, NADPH, ferredoxin and ferredoxin reductase (e.g., ferredoxin-NADP+ reductase) are mixed and incubated, for example, incubated at 20-40° C. (e.g., incubated at room temperature or 37° C.), to produce isoamylene. In some preferred embodiments, the method is used in the production of isoamylene in vitro.
In another aspect, the invention provides a method for producing isoamylene (e.g., 3-methyl-1-butene and/or 2-methyl-2-butene), comprising (a) culturing a cell expressing an exogenously introduced polypeptide having an amino acid sequence set forth in SEQ ID NO: 2; and (b) collecting isoamylene produced in step (a).
In some preferred embodiments, in step (a), the cell is cultured under conditions suitable for producing isoamylene (e.g., 3-methyl-1-butene and/or 2-methyl-2-butene). For example, in order to promote isoamylene production in a cell, the cell may be provided with one or more of the following substances: (1) a culture medium for retaining or promoting cell growth; (2) the substrate of the polypeptide according to the invention, DMAPP; (3) an electron transporter (e.g., ferredoxin), (4) an enzyme (e.g., ferredoxin reductase) needed for an electron transporter to transport electron; and (5) NADPH or NADH.
A variety of suitable culture media for culturing cells are well known by a person skilled in the art, and are commercially available.
In some preferred embodiments, the cell further expresses an electron transporter (e.g., ferredoxin) and/or an enzyme (e.g., ferredoxin reductase) needed for an electron transporter to transport electron. For example, the cell further expresses ferredoxin and ferredoxin reductase. In some preferred embodiments, the electron transporter and/or the enzyme needed for an electron transporter to transport electron is endogenous relative to the cell. In some preferred embodiments, the electron transporter and/or the enzyme needed for an electron transporter to transport electron is exogenous relative to the cell. For example, the ferredoxin and/or ferredoxin reductase may be endogenous or exogenous relative to the cell. In some preferred embodiments, the ferredoxin reductase is ferredoxin-NADP+ reductase (EC 1.18.1.2).
In some cases, the cell naturally expresses ferredoxin and ferredoxin reductase. In such a cell, it is not necessary to exogenously introduce a nucleic acid encoding ferredoxin and ferredoxin reductase. However, it is particularly preferred that in such a cell, a nucleic acid encoding ferredoxin and ferredoxin reductase is further introduced, to increase the expression of ferredoxin and ferredoxin reductase, thereby further enhancing the activity of the polypeptide according to the invention. In some cases, the cell does not express ferredoxin and ferredoxin reductase. In such a cell, it is particularly preferred that a nucleic acid encoding ferredoxin and ferredoxin reductase is introduced, so as to provide a high-efficient electron donor for the polypeptide according to the invention.
In some preferred embodiments, DMAPP may be added to a cell culture medium, so as to provide a substrate for the polypeptide according to the invention. In some preferred embodiments, DXP pathway is established in a cell to promote DMAPP synthesis in the cell, thereby providing a substrate for the polypeptide according to the invention. For example, one or more polypeptides involved in DXP pathway can be expressed in a cell to promote DMAPP synthesis in the cell, thereby providing a substrate for the polypeptide according to the invention
Therefore, in some preferred embodiments, the cell further expresses a polypeptide of DXP pathway. In some preferred embodiments, the polypeptide of DXP pathway is selected from the group consisting of 1-deoxy-D-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR; EC 1.1.1.267), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (MCT; EC 2.7.7.60), 4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol kinase (CMK; EC 2.7.1.148), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS; EC 4.6.1.12), 4-hydroxy-3-methyl-2-(E)-butenyl-diphosphate synthase (IspG; EC 1.17.7.1), and any combination thereof. In some preferred embodiments, the cell expresses one, two, three, four, five or six of said polypeptides of DXP pathways.
In some preferred embodiments, the polypeptide of DXP pathway is endogenous relative to the cell. In some preferred embodiments, the polypeptide of DXP pathway is exogenous relative to the cell.
In some preferred embodiments, the cell further expresses isopentenyl diphosphate isomerase (IDI; EC 5.3.3.2). In some preferred embodiments, the isopentenyl diphosphate isomerase is endogenous relative to the cell. In some preferred embodiments, the isopentenyl diphosphate isomerase is exogenous relative to the cell.
In some preferred embodiments, the cell is selected from the group consisting of a prokaryotic cell such as E. coli cell and bacillus cell (e.g., Bacillus alcalophilus, Bacillus subtilis), and a eukaryotic cell such as a yeast cell, an insect cell, a plant cell and an animal cell. However, it is particularly preferred that the cell is a prokaryotic cell, such as E. coli or Bacillus spp. or blue-green algae.
In some preferred embodiments, the method is used in biosynthesis of isoamylene (e.g., 3-methyl-1-butene and/or 2-methyl-2-butene).
In another aspect, the invention provides a method for preparing the polypeptide according to the invention, comprising (a) culturing a host cell comprising and expressing a nucleic acid encoding the polypeptide; and (b) collecting the polypeptide expressed by the cell.
A variety of host cells for protein expression are well known by a person skilled in the art, including, but not limited to a prokaryotic cell such as E. coli cell, and a eukaryotic cell such as a yeast cell, an insect cell, a plant cell and an animal cell (for example, a mammalian cell, such as a mouse cell and a human cell). It is particularly preferred that the host cell is E. coli. 
Definition and Explanation of Relevant Terms
In the invention, unless otherwise specified, the scientific and technical terms used herein have the meanings as generally understood by a person skilled in the art. Moreover, the relevant laboratory operating steps as used herein are the conventional steps widely used in the corresponding field. In addition, in order to understand the invention better, definitions and explanations are provided below for relevant terms.
As used herein, the term “HMBPP” refers to 4-hydroxy-3-methyl-but-2-enyl pyrophosphate, the structural formula of which is shown in the following Formula (I):

As used herein, the term “DMAPP” refers to dimethylallyl pyrophosphate, the structural formula of which is shown in the following Formula (II):

As used herein, the term “2M2B” refers to 2-methyl-2-butene, having a structural formula of
As used herein, the term “3M1B” refers to 3-methyl-1-butene, having a structural formula of
As used herein, the term “isoprene” refers to 2-methyl-1,3-butadiene, having a structural formula of

As used herein, the term “identity” refers to the match degree between two polypeptides or between two nucleic acids. When two sequences for comparison have the same base or amino acid monomer sub-unit at a certain site (e.g., each of two DNA molecules has an adenine at a certain site, or each of two polypeptides has a lysine at a certain site), the two molecules are identical at the site. The percent identity between two sequences is a function of the number of identical sites shared by the two sequences over the total number of sites for comparison ×100. For example, if 6 of 10 sites of two sequences are matched, the two sequences have an identity of 60%. For example, DNA sequences, CTGACT and CAGGTT, share an identity of 50% (3 of 6 sites are matched). Generally, the alignment of two sequences is conducted in a manner to produce maximum identity. Such alignment can be conducted by using a computer program such as Align program (DNAstar, Inc.) which is based on the method of Needleman, et al. (J. Mol. Biol. 48:443-453, 1970). The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
As used herein, the term “conservative substitution” refers to an amino acid substitution which would not disadvantageously affect or change the essential properties of a protein/polypeptide comprising an amino acid sequence. For example, a conservative substitution may be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions wherein an amino acid residue is substituted with another amino acid residue having a similar side chain, for example, a residue physically or functionally similar (such as, having similar size, shape, charge, chemical property including the capability of forming covalent bond or hydrogen bond, etc.) to the corresponding amino acid residue. The families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having alkaline side chains (for example, lysine, arginine and histidine), amino acids having acidic side chains (for example, aspartic acid and glutamic acid), amino acids having uncharged polar side chains (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having β-branched side chains (such as threonine, valine, isoleucine) and amino acids having aromatic side chains (for example, tyrosine, phenylalanine, tryptophan, histidine). Therefore, conservative substitution generally refers to substitution of a corresponding amino acid residue with another amino acid residue from the same side-chain family. Methods for identifying amino acid conservative substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).
In the application, it has been, demonstrated that IspH protein set forth in SEQ ID NO: 2 can convert HMBPP to isoprene. At the same time, methods for mutating a known polypeptide to obtain its mutant have been described in the prior art in detail, and are exemplarily described in the present application. For example, Examples of the description provide methods for preparing mutants of the IspH protein, and prepare 2 mutants (H131N and E133Q). In addition, methods for determining whether a polypeptide has an activity of converting HMBPP to isoprene are known by a person skilled in the art and are described in detail in the application. For example, Examples of the description provide methods for determining whether H131N and E133Q can use HMBPP as a substrate to produce isoprene, and it has been demonstrated that both H131N and E133Q have the activity of converting HMBPP to isoprene. Therefore, the methods according to the invention can be carried out repeatedly to prepare other mutants of the IspH protein which have the activity of converting HMBPP to isoprene. Therefore, the polypeptides according to the invention are not limited to IspH protein set forth in SEQ ID NO: 2 and its mutants H131N and E133Q, and intend to cover all the other mutants of IspH protein as long as they still retain the activity of converting HMBPP to isoprene.
As used herein, the expression “nucleic, acid/polypeptide is heterogeneous relative to a cell” means that the nucleic acid/polypeptide is not naturally present in the cell. That is, the cell in its natural state does not comprise or express the nucleic acid/polypeptide.
As used herein, the expression “nucleic acid/polypeptide is endogenous relative to a cell” means that the nucleic acid/polypeptide is naturally present in the cell. That is, the cell in its natural state comprises or expresses the nucleic acid/polypeptide.
As used herein, the expression “nucleic acid/polypeptide is exogenous relative to cell” means that the nucleic acid/polypeptide is exogenously introduced into the cell artificially. It should be understood that such a nucleic acid/polypeptide may not be naturally present in the cell (i.e., is heterogenous relative to the cell), and is used to introduce a heterogenous nucleic acid/polypeptide into a cell; or may also be identical to an endogenous nucleic acid/polypeptide that is naturally present in the cell, and is used to increase the copy number or expression of the endogenous nucleic acid/polypeptide in the cell.
As used herein, the term “electron transporter” refers to a protein involved in electron transport in an electron transport chain, in general, an electron transporter can not only accept electrons as an electron acceptor, but also provide electrons as an electron donor, and therefore can achieve electron transport. Such electron transporters are well known by a person skilled in the art, including, but not limited to, ferredoxin and flavodoxin. In addition, the involvement of an oxido-reductase (which is also called an enzyme needed for an electron transporter to transport electron) is generally needed during electron transport by an electron transporter. For example, the involvement of ferredoxin reductase is generally needed in electron transport of ferredoxin; the involvement of flavodoxin reductase is generally needed in electron transport of flavodoxin. An enzyme needed for an electron transporter to transport electron is also well known by a person skilled in the art, for example, including, but not limited to ferredoxin reductase and flavodoxin reductase.
As used herein, the term “ferredoxin reductase” refers to an enzyme capable of catalyzing oxidation-reduction reactions of ferredoxin, including two types:
(1) ferredoxin-NADP+ reductase, which has an IntEnz accession number of EC 1.18.1.2, and can catalyze the following reaction:
2 ferredoxin in reduced form+NADP++H+2 ferredoxin in oxidized form+NADPH; and
(2) ferredoxin-NAD+ reductase, which has an IntEnz accession number of EC 1.18.1.3, and can catalyze the following reaction:
2 ferredoxin in reduced form+NAD++H+2 ferredoxin in oxidized form+NADH.
In the invention, ferredoxin reductase is preferably ferredoxin-NADP+ reductase (EC 1.18.1.2).
As used herein, the term “a polypeptide of DXP pathway” refers to a polypeptide involved in DXP pathway. DXP pathway refers to a pathway of synthesizing DMAPP from pyruvate and glyceraldehyde 3-phosphate as raw materials, comprising the following steps: (1) producing 1-deoxy-D-xylulose 5-phosphate (DXP) by condensation of pyruvate and glyceraldehyde 3-phosphate; (2) converting DXP to 2-C-methyl-D-erythritol 4-phosphate (MEP); (3) converting MEP to 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol (CDP-ME); (4) converting CDP-ME to 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol 2-phosphate (CDP-ME2P); (5) converting CDP-ME2P to 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP); (6) converting MECDP to HMBPP; and (7) converting HMBPP to DMAPP and IPP. The detailed description of DXP pathway may be found in, for example, Tomohisa Kuzuyama, Biosci. Biotechnol. Biochem., 66(8), 1619-1627, 2002; Thomas D. Sharkey et al., Plant Physiology, February 2005, Vol. 137, pp. 700-712; and U.S. Pat. No. 8,507,235B2.
The polypeptides involved in DXP pathway include, but are not limited to 1-deoxy-D-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR; EC 1.1.1.267), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (MCT; EC 2.7.7.60), 4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol kinase (CMK; EC 2.7.1.148), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS; EC 4.6.1.12), 4-hydroxy-3-methyl-2-(E)-butenyl-diphosphate synthase (IspG; EC 1.17.7.1). The detailed description of these polypeptides (enzymes) can be found in the public database IntEnz (http://www.ebi.ac.uk/intenz/).
As used herein, the term “isoprene synthase (IspS)” refers to an enzyme capable of using DMAPP as a substrate to produce isoprene, which has an IntEnz accession number of EC 4.2.3.27.
As used herein, the term “isopentenyl diphosphate isomerase (IDI)” refers to an enzyme capable of catalyzing the isomerization between DMAPP and IPP, also called IPP isomerase, which has an IntEnz accession number of EC 5.3.3.2.